The invention relates to novel mimetics of the tetrasaccharides sialyl-Lewis-X (SLeX) and sialyl-Lewis-A (SLeA) with improved action as inhibitors of cell adhesion, to a process for the preparation of these compounds and to their use as pharmacological active compounds and diagnostic agents.
The circulation of blood cells, for example leukocytes, neutrophils, granulocytes and monocytes, is, at the molecular level, a highly complex multistage process of which only individual steps are known (for a review see: Springer, Cell 76:301-314 (1994)). Recent research has shown that both localization of neutrophils and monocytes at foci of inflammation and lymphocyte recirculation, which is crucial in immune monitoring, respond to very similar molecular mechanisms. Thus, in acute and chronic inflammatory processes leukocytes adhere to endothelial cells and migrate to the focus of inflammation and into the secondary lymphatic organs. This process involves numerous specific signal molecules, for example interleukins, leukotrienes and tumor necrosis factor (TNF), G-protein coupled receptors and, in particular, tissue-specific cell adhesion molecules, which precisely control immune cell and endothelial cell recognition. The most important adhesion molecules involved in this process, designated below as receptors, include the selectins (E-, P- and L-selectins), integrins and the members of the immunoglobulin superfamily.
Adhesion of leukocytes to endothelial cells is mediated by selectin receptors in the initial phase of inflammatory processes, and is a natural and necessary immune response to various inflammatory stimuli and to vascular tissue damage. The course of a variety of acute and chronic disorders, such as rheumatism, reperfusion injuries such as myocardial ischemia/infarct (MI), acute pneumonia following surgery, traumatic shock and stroke, psoriasis, dermatitis, ARDS (adult respiratory distress syndrome) and restenosis following surgical intervention (for example angioplasty and by-pass operations) is, however, adversely affected by excessive leukocyte adhesion and infiltration into affected tissue. Controlling this adhesion process at a very early stage of inflammation is, therefore, a highly attractive and generally applicable concept for the pharmacological control of inflammatory disorders.
The tetrasaccharides sialyl-Lewis-X (SLeX) and sialyl-Lewis-A (SLeA), which occur as substructures of glycosphingolipids and glycoproteins on cell membranes, can function as ligands for all three selectin receptors. A series of glycoproteins, mucins and glycolipids are known to be suitable endogenous ligands for the selecting. These include: Mucosal Vascular Addressin MadCAM-1 (Berg et al., Nature 366:695 (1993)) and Sialomucin CD34 (Baumhuter et al., Science 262:436 (1993)) for L-selectin: O-linked polylactosamine-sialomucin PSGL-1 on human neutrophils for P-selectin (Moore et al., J.Biol.Chem. 269:23318 (1994); and N-linked sialoglycoproteins of the ESL-1 type for E-selectin (Vestweber et al., Cell Biol. 121:449 (1993)).
The specificity of these and other potential ligands for selectins in vivo has not yet been elucidated. The tetrasaccharides SLeX and SLeA represent only a substructure of the substantially more complex structures of endogenous selectin ligands and, because of their similar affinity for selecting, cannot alone account for receptor binding specificity. Due to the structural complexity of SLeX and SLeA, the use of simpler, structurally modified mimetics as antagonists for modulating or suppressing excessive leukocyte adhesion is a promising therapeutic starting point for a strategy for alleviating or healing the above-mentioned disorders mentioned.
SLeX has already been used successfully in animal experiments to protect against P-selectin-dependent lung damage (Mulligan et al., Nature 364:149 (1993)) and against myocardial reperfusion injuries (Buerke et al., J.Clin.Invest. 93:1140 (1994)). In initial clinical trials against acute pneumonia, the compound was employed in a dose of 1-2 grams per day per patient (report by Cytel Corp./La Jolla (Calif.) at the 2nd Glycotechnology Meeting/CHI in La Jolla/USA on May 16-18, 1994).
Some publications and patent applications have also reported efforts to obtain more potent antagonists by structural variation of the ligand. The aim of such work is to provide more effective antagonists that potentially would also be suitable for use in vivo at a relatively low dose. Variation of the fucose and neuraminic acid units regarded as crucial for the structure-activity relationship (Brandley et al., Glycobiology 3:633 (1993); Yoshida et al., Glycoconjugate J. 10:3 (1993)), did not, however, afford significantly improved inhibition. Only when the glucosamine unit was varied (replacement of GlcNAc by glucose and azido groups and amino groups in position 2 of GlcNAc) was significantly increased affinity for the E-selectin receptor achieved. By contrast, improved binding of the P-selectin receptor was not achieved.
In general, all previous successes have been limited to improving the binding affinity of SLeX and SLeA derivatives for the E-selectin receptor, since at inhibitor concentrations of about 1 mM only weak inhibitory effects with the P-selectin receptor have been found (Nelson et al., J.Clin.Invest. 91:1157 (1993)). The binding affinities of modified SLeX/A structures for selecting has been reviewed. See Pharmacochem. Libr. 20 (1993))(Trends in Drug Research), pp. 33-40.
In addition to their low affinity for selectin binding, these compounds all contain at least one unstable glycosidic linkage, which severely restricts their oral availability as active compounds. This instability also greatly limits the synthesis of various derivatives, since the hydrolytic lability of the glycosidic linkage limits the available reaction conditions. A number of strategies for synthesizing mimetics have been developed to obtain an increase in hydrolytic stability.
For example, stability has been increased by attaching the side chain via a Cxe2x80x94C bond to the C-4 carbon of fucose (Floyd et al., Tetrahedron Asymmetry 5:2061 (1994). 
In this case, however, the linkage to C-4 of fucose caused the orientation of the side chain to differ from that of the natural ligand, and only low affinity for selectin binding was observed.
Use of carbocyclic carbohydrate analogs where the side chain linkage is through a Cxe2x80x94C bond to C-1 would give a conformation similar to that of the natural ligand that also would be stable against degradation. Several carbocyclic carbohydrate analogs of monosaccharide units have been prepared. Thus, for example, activated monosaccharides have been reacted with nitromethane (Gross, Tetrahedron 47:6113 (1991)), allylsilane (Kishi et al., J. Am. Chem. Soc. 104:4976-4978 (1982)), and olefins (Levy et al., Tetrahedron Asymmetry 5:2265-2268 (1994)). The functionality introduced into these monosaccharides makes them suitable as a unit for further coupling operations.
Use of a carbocyclic analog as a building block for selectin antagonists has been shown to lead to a mimetic with affinity for selectins. It was possible at the same time, by reacting a fucose unit with allylsilane, to synthesize a specific C-glycosidic unit (1) with an xcex1-orientation at the side chain (WO 95/04751). Selectivities in the allylation are high, with xcex1/xcex2=14/1, but scale-up of the reaction is difficult due to the conditions employed, particularly the need to use 10 equivalents of volatile and highly corrosive trimethylsilyl triflate. Similarly, chromatographic purification of the product is required, although it is not possible to separate the xcex1/xcex2 mixture. 
The terminal acid function of the side chain can be used to synthesize glycopeptide analogs, for example (2). These analogs have IC50 values of about 1 mM, but are unstable to proteolytic degradation. Consequently, despite stabilization of the sugar unit by the C-glycoside, the oral availability of these compounds continues to be severely restricted. A further disadvantage is the presence of the unwanted xcex2 compound, which cannot be separated. These xcex2 derivatives show no activity, due to the side chain having the wrong orientation, as demonstrated by corresponding model calculations. 
The C-glycosidic unit (1) has also been used in an analogous manner, for synthesizing various other mimetics intended to imitate the active conformation of SleX. The compounds tested, for example (3), however, exhibited IC50 values of 10-20 mM, higher by a factor of 10-20 than that of the natural ligand SLeX (Wong, et al., J. Am. Chem. Soc. 117:5395(1995)). 
Use of a substituted allylsilane allowed preparation of a C-glycoside. 
By linking to triterpenoid acid derivatives (Betulinic Acid: WO 95/04526; Glycyrrhetinic Acid: WO 94/24145), mimetics such as (4a) were prepared. These were intended to have a multi-medicament capacity and were tested in a variety of test systems (inhibition of 5-lipoxygenase, antimetastatic action, P-selectin inhibition). In this case an IC50 of 0.75 mM for binding to P-selectin was obtained. It should be noted, however, that the triterpenoid acid alone displayed an IC50 of 0.125 mM. 
Preparation of the C-glycoside unit (4) likewise required a complex chromatographic purification, where the xcex2 derivative was separated only with difficulty. Additionally, stability of the C-glycoside was limited due to the reactive allylic chloride group.
In addition to the preparative problems described above (such as complex chromatographic purification, multiple synthetic steps due to protective-group strategy, xcex1/xcex2 mixtures) the aforementioned C-glycoside mimetics displayed a selectin binding affinity too low for effective inhibition of adhesion processes. In addition to stability problems with a fucose mimetic, the low affinity of the aforementioned derivatives is also due to the need for correct orientation and fixed conformation of side chains that is important for selectin binding, which the derivatives apparently do not satisfy.
According to L. A. Lasky, negatively charged sialic acid (or a negatively charged sulfonic acid group) is an absolute requirement for binding to selectins. Studies to determine potential binding sites have already been carried out using the recently elucidated crystal structure of E-selectin. Possible binding sites for the sialic acid function have been proposed, including the two lysines K111 and K113 (Bajorath et al., Biochemistry 33:1332 (1994)) and Arg 97, Lys 111 and Lys 113 (Structural Biology 1:140 (1994)).
It is apparent, therefore, that readily prepared, stable, low molecular weight mimetics of sialyl-Lewis-X and sialyl-Lewis-A structures possessing significantly enhanced affinity for selectins are greatly to be desired. It is also greatly desirable that these mimetics possess a structure that makes them suitable for oral administration.
It is therefore an object of the present invention to provide stable, low molecular mass mimetics of sialyl-Lewis-X and sialyl-Lewis-A structures, respectively, whose constitution and configuration possess a significantly enhanced affinity for selecting, which are easier to obtain by synthesis than oligosaccharides and whose structure makes them suitable as pharmaceuticals with potential for oral availability.
These and other objects of the invention are achieved by providing a compound of the formula I 
in which n is 1 or 2; R1 is xe2x80x94H, xe2x80x94CH2OH or xe2x80x94CH3; R2 and R3 independently of one another are xe2x80x94H or xe2x80x94OH; R4 and R5 independently of one another are xe2x80x94H, xe2x80x94OHxe2x80x94, -alkyl, xe2x80x94O-alkyl, xe2x80x94S-alkyl, xe2x80x94NH2, xe2x80x94NH-alkyl, xe2x80x94N(alkyl)2, xe2x80x94NH-aryl, xe2x80x94N(aryl)2, xe2x80x94OSO3H, xe2x80x94(CH2)rxe2x80x94COOH, xe2x80x94(CH2)rxe2x80x94COO-alkyl, xe2x80x94(CH2)rCH(COO-alkyl)2, xe2x80x94(CH2)rCH(COOH)2, xe2x80x94(CH2)rxe2x80x94NH2, where r is an integer from zero to ten, or R4 and R5 together form a double bond or an epoxide ring; A, B, D, E and G independently are CR6, CR7, CR8, CR9, CR10 or nitrogen, provided that only one of the variables A, B, D, E and G may be nitrogen; R6, R7, R8, R9 and R10 independently of one another are xe2x80x94H, -alkyl, xe2x80x94OH, xe2x80x94O-alkyl, xe2x80x94NH2, xe2x80x94NH-alkyl, xe2x80x94N(alkyl)2, xe2x80x94NH-aryl, xe2x80x94N(aryl)2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94COO-alkyl, xe2x80x94COxe2x80x94NH2, xe2x80x94COOH, xe2x80x94OSO3H, 4-hydroxypiperidin-4-yl, xe2x80x94(CH2)mxe2x80x94COOH, xe2x80x94(CH2)mCOO-alkyl, xe2x80x94(CH2)mxe2x80x94CH(COO-alkyl)2, xe2x80x94(CH,)mxe2x80x94CH(COOH)2, where m is an integer from zero to ten, xe2x80x94(CH2)pxe2x80x94NH2, where p is an integer from one to ten, a group of the formula II, III, IV, V, VI, or VII, 
where X1 and X2 independently are H or an oligopeptide, or two of R6, R7, R8, R9 or R10, provided that they are adjacent, together form a carboxymethyl-substituted imidazole ring or a crown ether ring.
In accordance with one aspect of the invention, R1, R2, and R3 have the same absolute configuration as in L-fucose, D-mannose, or D-ribose. In a preferred embodiment R4 and R5 are both H, or R4 and R5 together from a double bond. In another preferred embodiment, one of the variables A, B, G, E or D is selected from the group consisting of Cxe2x80x94COOH, Cxe2x80x94CH2xe2x80x94COOH, Cxe2x80x94CH(COOH)2, and NH2, and all of the other of these variables are Cxe2x80x94H. In yet another preferred embodiment, the variables A, D and G are Cxe2x80x94H, the variable B is nitrogen and the variable E is Cxe2x80x94CH2xe2x80x94COOH. In still another preferred embodiment, A,B, G and E are Cxe2x80x94H and D is CR8.
In accordance with another aspect of the invention, there is provided compounds where R8 is selected from the group consisting of II, III, IV, V, VI, and VII. In a preferred embodiment, R8 is VII, and X1 and X2 independently of one another are H or an oligopeptide. In other preferred embodiments, R8 is II or IV. In another preferred embodiment, the oligopeptide comprises L-amino acids, and preferably comprises the sequence arg-gly-asp-ser.
In accordance with yet another aspect of the invention, there are provided compounds where one of the variables A,B, G, E and D is CR9 and all of the other of these variables are Cxe2x80x94H. In a preferred embodiment, R9 is a 4-hydroxypiperidin-4-yl group.
In accordance with still another aspect of the invention there are provided compounds where E and G together form a substituted imidazole ring and A, B and D are Cxe2x80x94H.
In accordance with a still further aspect of the invention there are provided compounds where A and B are Cxe2x80x94H, G and E are Cxe2x80x94OH and D is Cxe2x80x94COOH.
In accordance with another aspect of the invention there is provided a method for preparing a compound of the type described above, comprising reacting a compound of the formula VIII, 
where R1, R2 and R3 are present in protected or unprotected form, with a compound of the formula IX, 
where X is halogen, and G and E are linked by a single or a double bond, in the presence of a transition metal catalyst. In one preferred embodiment, R1, R2 and R3 are unprotected. In another preferred embodiment, the transition metal catalyst is a palladium catalyst.
In accordance with yet another aspect of the invention there is provided a pharmaceutical composition comprising a compound as set forth above, together with a pharmaceutically acceptable excipient.
In accordance with still aspect of the invention there is provided a method of treating a disease associated with excessive selectin-mediated cell adhesion, comprising administering a pharmaceutical composition as set forth above to a patient suffering from said disease.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The present invention provides novel glycomimetics that bind to both E- and P-selectin, and that possess antiinflammatory activity. These glycomimetics have structures that are variants of the natural ligands sialyl Lewis X and sialyl Lewis A, but have enhanced stability against degradation in vitro and in vivo. Moreover, the glycomimetics are readily prepared in quantity by organic synthesis methods well known in the art. The invention also includes pharmaceutical compositions comprising an effective amount of at least one of the glycomimetics of the invention in combination with a pharmaceutically acceptable sterile vehicle, as described, for example, in Remingtons"" Pharmaceutical Sciences; Drug Receptors and Receptor Theory, 18th ed., Mack Publishing Co., Easton, Pa. (1990). Compounds according to the present invention, and their physiologically tolerated salts, are suitable for use as medicaments in mammals, especially humans.
The glycomimetics of the present invention may generally be represented by the formula I 
In this formula, R1 is xe2x80x94H, xe2x80x94CH2OH or xe2x80x94CH3, R2 and R3 independently of one another are xe2x80x94H or xe2x80x94OH, and n is 1 or 2. R4 and R5 independently of one another are xe2x80x94H, xe2x80x94OH-, -alkyl, xe2x80x94O-alkyl, xe2x80x94S-alkyl, xe2x80x94NH2, xe2x80x94NH-alkyl, xe2x80x94N(alkyl)21, xe2x80x94NH-aryl, xe2x80x94N(aryl)2, xe2x80x94OSO3H, xe2x80x94(CH2)rxe2x80x94COOH, xe2x80x94(CH2)rxe2x80x94COO-alkyl, xe2x80x94(CH2)rCH(COO-alkyl)2, xe2x80x94(CH2)rCH(COOH)2,xe2x80x94or (CH2)rxe2x80x94NH2. r is an integer from zero to ten. R4 and R5 may also together form a double bond or an epoxide ring.
A, B, D, E and G are CR6, CR7, CR8, CR9, CR10 or nitrogen, provided that in each case only one of the variables A, B, D, E and G is nitrogen. R6, R7, R8, R9 and R10 independently of one another are xe2x80x94H, -alkyl, xe2x80x94OH, xe2x80x94O-alkyl, xe2x80x94NH2, xe2x80x94NH-alkyl, xe2x80x94N(alkyl)2, xe2x80x94NH-aryl, xe2x80x94N(aryl)2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94COO-alkyl, xe2x80x94COxe2x80x94NH2, xe2x80x94COOH, xe2x80x94OSO3H, 4-hydroxypiperidin-4-yl, xe2x80x94(CH2)mxe2x80x94COOH, xe2x80x94(CH2)mCOO-alkyl, xe2x80x94(CH2)mxe2x80x94CH(COO-alkyl)2, xe2x80x94(CH2)mxe2x80x94 or CH(COOH)2, where m is an integer from zero to ten. R6, R7, R8, R9 and R10 also can be xe2x80x94(CH2)pxe2x80x94NH2, where p is an integer from one to ten. Alternatively, R6, R7, R8, R9 and R10 independently of one another can be selected from the group consisting of moieties having the structures II, III, IV, V, VI, or VII. In formula V, q is an integer from one to ten. In formula VII, X1 and X2 independently of one another are H or an oligopeptide. Alternatively, X1 and X2 independently are two of the variables R6, R7, R8, R9 and R10, that, provided they are adjacent, together form a carboxymethyl-substituted imidazole ring or a crown ether ring, with the other variables being as defined above. 
In a preferred embodiment of the invention, in a compound of the formula I, one of the variables A,B, G, E and D is Cxe2x80x94COOH and the other variables are Cxe2x80x94H. Examples of such embodiments are compounds having the formulae 16, 21, 22a, 23, or 29 
In another preferred embodiment one of the variables A,B, G, E and D is Cxe2x80x94CH2xe2x80x94COOH and the other variables are Cxe2x80x94H. Examples of these embodiments are compounds 28 and 28a: 
In another preferred embodiment, one of the variables A,B, G, E and D is Cxe2x80x94CHxe2x80x94(COOH)2 and the other variables are Cxe2x80x94H. An example of such an embodiment is compound 24: 
In yet another preferred embodiment, one of the variables A,B, G, E and D is Cxe2x80x94NH2 and the other variables are Cxe2x80x94H. Examples of these embodiments are compounds 27 and 30: 
In still another preferred embodiment, the variables A, D and G are Cxe2x80x94H, B is nitrogen and E is Cxe2x80x94CH2xe2x80x94COOH, for example, compound 26: 
Alternatively, A, B, G and E are Cxe2x80x94H and D is CR8. R8 is preferably a moiety having the formula II 
An example of such an embodiment is compound 20: 
Alternatively, R8 is a group having the formula III 
An example of such an embodiment is compound 18: 
In another alternative, R8 is a moiety having the formula IV 
An example of such an embodiment is compound 25: 
In still another alternative, R8is a moiety having the formula V, where q is an integer from one to ten, 
An example of such an embodiment is compound 34: 
In another alternative, R8 is a group of the formula VI: 
An example of such an embodiment is compound 33: 
R8 may also be a group having the formula VII 
where X1 and X2 independently of one another are H or an oligopeptide. An example of such an embodiment is compound 36: 
In other preferred embodiments, one of the variables A,B, G, E and D is CR9 and the other variables are Cxe2x80x94H. Preferably, R9 is a 4-hydroxypiperidin-4-yl group, for example compound 32: 
Alternatively, E and G together form a substituted imidazole ring and A, B and D are Cxe2x80x94H, for example compound 31: 
In yet another embodiment, A and B are Cxe2x80x94H, G and E are Cxe2x80x94OH and the D is Cxe2x80x94COOH, for example compound 37: 
Glycomimetics according to the present invention may be prepared by reacting a compound of the formula VIII 
where R1, R2 and R3, and n are as defined above, with a compound of the formula IX 
In this reaction, R1, R2 and R3 may be present in protected or unprotected form, and A, B, D, E and G are as defined above. X is a halogen atom, and the bond between G and E can also be a single bond (i.e. IX is a 1,3-cyclohexadienyl derivative). The reaction proceeds under transition metal catalysis, where the transition metal preferably is palladium. Following the coupling reaction, the reaction product may be transformed into the desired final product using methods that are well known to those skilled in the art of organic synthesis. Any protecting groups used in the coupling reaction may be removed by methods that are well known in the art. The compounds according to the present invention can be purified by well known methods, such as chromatography, recrystallization, etc.
The glycomimetics of the present invention are therefore useful in pharmaceuticals for therapy or prophylaxis of diseases associated with excessive selectin-mediated cell adhesion. These pharmaceuticals are particularly suitable for the treatment of acute and chronic inflammation that is characterized pathophysiologically by a disruption of cell circulation, for example of lymphocytes, monocytes and neutrophil granulocyte circulation. Such inflammation is found in: autoimmune diseases such as acute polyarthritis, rheumatoid arthritis and insulin-dependent diabetes mellitus (IDDM); acute and chronic transplant rejections; shock lung (ARDS, adult respiratory distress syndrome); inflammatory and allergic skin diseases, for example psoriasis and contact eczema; cardiovascular disorders such as myocardial infarct; reperfusion injuries after thrombolysis, angioplasty or by-pass operations; septic shock; and systemic shock. A further indication is the treatment of metastatic tumors, since tumor cells carry surface antigens possessing both sialyl-Lewis-X and sialyl-Lewis-A structures as recognition epitopes. It is also possible to use these pharmaceuticals, which are stable in the acidic medium of the stomach, for antiadhesive therapy of Helicobacter pylori and related microorganisms, alone or in combination with antibiotics. In addition, the pharmaceuticals may be used for therapy of the cerebral form of malaria.
The pharmaceuticals according to the invention may be administered intravenously, orally or parenterally or as implants, and rectal application is also possible. Suitable solid or liquid pharmaceutical preparation forms are well known in the art, and include, for example, granules, powders, plain tablets, film-coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, aerosols, drops and injectable solutions in ampule form. Formulations with protracted release of active compound may also be used. These formulations customarily are prepared using excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, glidants or lubricants, flavorings, sweeteners or solubilizers. Frequently used excipients or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactoprotein, gelatin, starch, vitamins, cellulose and their derivatives, animal and vegetable oils, poly-ethylene glycols and solvents, for instance sterile water, alcohols, glycerol and polyhydric alcohols. The pharmaceutical preparations are preferably prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories.
For patient treatment, different daily doses are required depending on the efficacy of the compound, the mode of application, the nature and severity of the disease and the age and body weight of the patient. Under certain circumstances, however, higher or lower daily doses may also be appropriate. Determination of an appropriate dosage regimen for a patient is routine for those skilled in the art of prescribing pharmaceuticals. The daily dose additionally may depend on the number of receptors expressed during the course of the disease. In the initial stage of the disease only a few receptors may be expressed on the cell surface and, accordingly, the daily administered dose is lower than in the case of patients who are severely ill. Once a daily dose is determined, it can be administered either in a single administration in the form of an individual dose unit, or in a number of small dose units, or by multiple administration of subdivided doses at defined intervals. The pharmaceuticals according to the invention are produced by bringing a compound according to the present invention into the or an appropriate administration form with customary excipients and, if desired, additives and/or auxiliaries.
Synthesis of Compounds of the Formula I
The synthesis of compounds of Formula I is illustrated below for the compound 8. Those skilled in the art will recognize that the synthetic scheme described below may be modified by methods that are well known in the art to provide all of the compounds corresponding to Formula I.
The synthesis of the pure xcex1-C-glycoside 8 was carried out in only 3 stages with an overall yield of 72, by the reaction scheme shown below. 
Thus, L-fucose 5 was acetylated with acetic anhydride/pyridine to provide the tetra acetate (95-98%), which was then treated, under BF3catalysis, with allylsilane. The resulting C-glycoside 7 (92%, xcex1/xcex2 ratio 10:1), was quantitatively deacetylated, and purified by recrystallization. By appropriate choice of the recrystallization conditions it was possible to obtain the xcex1 derivative 8 in pure form, (i.e. without additional amounts of the corresponding xcex2 derivative), in contrast to known processes that failed to remove the contaminating xcex2 isomer. The recrystallization is the only purification step in this sequence.
The allyl side chain of the C-glycoside 8 can then be functionalized without introduction of additional protective groups. Alternatively, the perbenzylated compound 8a may be used for further derivatization. 
A reaction that is particularly suitable for this derivatization is the transition metal-catalyzed Cxe2x80x94C linkage of a protected or unprotected C-allyl glycoside (e.g. 8 or 8a) with a substituted or unsubstituted aryl or heteroaryl halide/triflate (the so-called Heck reaction, see e.g. Larock et al., J. Org. Chem. 56:2615 (1991). This reaction also may be carried out using a 1,3-cyclohexadienyl halide or triflate in the coupling reaction, as described below in Example 27, for example.
The preferred catalyst for this reaction is a Pdxc2x0 compound. Examples of suitable catalysts include Pd(PPh3)4, Pd(OAc)2, and Pd(dba)2, where (dba) is dibenzylacetone. The catalyst may also be prepared in situ by addition of a reducing agent to a PdII salt, for example by adding P(Ph)3 to Pd(OAc)2.
The synthesis generally leads in a few steps to the pharmacologically relevant end compound in good to very good yields. The products of the Heck reaction can frequently be isolated by extraction or reprecipitation without further purification by chromatography.
The double bond may also easily be used for further derivatization. Examples of suitable derivatizations include: hydrogenation; halogenation; Diels-Alder reactions; epoxidation (Jacobsen et al., J. Am. Chem. Soc. 112:2801 (1990)); and hydroxylation (Sharpless et al., J. Am. Chem. Soc. 116:1278 (1994). These and other functionalization reactions well known to the skilled artisan provide efficient and simple access to modified derivatives. For example, the Heck reaction of Boc-4-iodophenylalanine (BOC=tbutoxycarbonyl) with the allyl fucoside gives the corresponding neoglycoamino acid, which may be employed successfully both in solid-phase and liquid phase peptide synthesis for the preparation of modified peptides.
The reactions described above can be applied analogously to other C-glycoside units, for example compounds 9 and 9a, which can be prepared from D-mannose, or compounds 10 and 10a, which can be prepared from ribose. 
The process according to the present invention can also be used to prepare, in corresponding fashion using techniques well known in the art, derivatives of L-galactose, L-rhamnose, and glucose.
Primary Assays to Investigate the Action of the Compounds According to the Present Invention on Cell Adhesion to Recombinant, Soluble Selectin Fusion Proteins.
To test the efficacy of the compounds according to the invention on the interaction between E- and P-selectins (former nomenclature ELAM-1 and GMP-140, respectively) with their ligands, assay that are specific for either E- or P-selectin interactions are used. In these assays, the ligands may be supplied in their natural form as surface structures on promyelocytic HL60 cells. Since HL60 cells contain ligands and adhesion molecules of very different specificity, the desired specificity of the assay must therefore be provided by the binding component. Preferably, the binding components used are recombinant, soluble, fusion proteins formed from the extracytoplasmatic domains of E- or P-selectin, respectively, and the constant region of human immunoglobulin of the IgG1 subclass. Preferably, a non-selectin binding fusion protein is used as a negative control. An example of a suitable negative control is a fusion protein formed between the extracytoplasmic domain of CD4 and the IgG constant region.
These fusion proteins are then used in a cell adhesion assay in which COS cells transfected with the recombinant fusion proteins are first bound to the surface of a microtiter plate. The compound to be tested is added to the wells of the microtiter platein varying concentrations, followed by HL60 cells. After a defined period of time, the wells of the microtiter plate are washed and the HL60 cells adhering to the plate are counted. In a preferred embodiment, the HL60 cells are prelabeled with a fluorescent dye to facilitate cell counting. The number of cells adhering to the plate allows quantification of the inhibitory properties of the compound under test. Methods for assaying cell adhesion inhibition are described in more detail infra.
Leukocyte Adhesionxe2x80x94Testing the Efficacy of the Novel Compounds in vivo
In inflammatory processes and other states which activate the cytokines, the destruction of tissue by inward migrating leukocytes or leukocytes which block the microcirculation plays a critical part. The first phase, which is critical for the subsequent disease process, is activation of leukocytes within the bloodstream, especially in the pre- and postcapillary region. The leukocytes leave the axial flow of the blood, and initially attach to the inner vascular wall, i.e. to the vascular endothelium. All subsequent leukocyte effects, i.e. the active migration through the vascular wall and the subsequent oriented migration in the tissue, are follow-on reactions (Harlan, Blood 65, 513-525, 1985).
This receptor-mediated interaction of leukocytes and endothelial cells is regarded as an initial sign of the inflammatory process. In addition to the adhesion molecules already physiologically expressed, under the action of inflammatory mediators (leukotrienes, platelet activating factor) and cytokines (TNF-alpha, interleukins), a temporally graded, massive expression of adhesion molecules takes place on the cells. They are at present divided into three groups: the immunoglobulin gene superfamily, the integrins, and the selecting. Whereas adhesion takes place between molecules of the Ig gene superfamily and the protein-protein bonds, lectin-carbohydrate bonds are predominant in the interaction of selectins (Springer, Nature 346:425 (1990); Hughes, Scrips Magazine 6:30 (1993); Springer, Cell 76:301 (1994)).
The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.